Enzymes of hydrogen metabolism in Pyrococcus furiosus.

The genome of Pyrococcus furiosus contains the putative mbhABCDEFGHIJKLMN operon for a 14-subunit transmembrane complex associated with a Ni-Fe hydrogenase. Ten ORFs (mbhA-I and mbhM) encode hydrophobic, membrane-spanning subunits. Four ORFs (mbhJKL and mbhN) encode putative soluble proteins. Two of these correspond to the canonical small and large subunit of Ni-Fe hydrogenase, however, the small subunit can coordinate only a single iron-sulfur cluster, corresponding to the proximal [4Fe-4S] cubane. The structural genes for the small and the large subunits, mbhJ and mbhL, are separated in the genome by a third ORF, mbhK, encoding a protein of unknown function without Fe/S binding. The fourth ORF, mbhN, encodes a 2[4Fe-4S] protein. With P. furiosus soluble [4Fe-4S] ferredoxin as the electron donor the membranes produce H2, and this activity is retained in an extracted core complex of the mbh operon when solubilized and partially purified under mild conditions. The properties of this membrane-bound hydrogenase are unique. It is rather resistant to inhibition by carbon monoxide. It also exhibits an extremely high ratio of H2 evolution to H2 uptake activity compared with other hydrogenases. The activity is sensitive to inhibition by dicyclohexylcarbodiimide, an inhibitor of NADH dehydrogenase (complex I). EPR of the reduced core complex is characteristic for interacting iron-sulfur clusters with Em approximately -0.33 V. The genome contains a second putative operon, mbxABCDFGHH'MJKLN, for a multisubunit transmembrane complex with strong homology to the mbh operon, however, with a highly unusual putative binding motif for the Ni-Fe-cluster in the large hydrogenase subunit. Kinetic studies of membrane-bound hydrogenase, soluble hydrogenase and sulfide dehydrogenase activities allow the formulation of a comprehensive working hypothesis of H2 metabolism in P. furiosus in terms of three pools of reducing equivalents (ferredoxin, NADPH, H2) connected by devices for transduction, transfer, recovery and safety-valving of energy.

Novel structure and redox chemistry of the prosthetic groups of the iron-sulfur flavoprotein sulfide dehydrogenase from Pyrococcus furiosus; evidence for a [2Fe-2S] cluster with Asp(Cys)3 ligands.

The consecutive structural genes for the iron-sulfur flavoenzyme sulfide dehydrogenase, sudB and sudA, have been identified in the genome of Pyrococcus furiosus. The translated sequences encode a heterodimeric protein with an alpha-subunit, SudA, of 52598 Da and a beta-subunit, SudB, of 30686 Da. The alpha-subunit carries a FAD, a putative nucleotide binding site for NADPH, and a [2Fe-2S]2+,+ prosthetic group. The latter exhibit EPR g-values, 2.035, 1.908, 1.786, and reduction potential, Em,8 = +80 mV, reminiscent of Rieske-type clusters; however, comparative sequence analysis indicates that this cluster is coordinated by a novel motif of one Asp and three Cys ligands. The motif is not only found in the genome of hyperthermophilic archaea and hyperthermophilic bacteria, but also in that of mesophilic Treponema pallidum. The beta-subunit of sulfide dehydrogenase contains another FAD, another putative binding site for NADPH, a [3Fe-4S]+,0 cluster, and a [4Fe-4S]2+,+ cluster. The 3Fe cluster has an unusually high reduction potential, Em,8 = +230 mV. The reduced 4Fe cluster exhibits a complex EPR signal, presumably resulting from magnetic interaction of its S = 1/2 spin with the S=2 spin of the reduced 3Fe cluster. The 4Fe cluster can be reduced with deazaflavin/EDTA/light but not with sodium dithionite; however, it is readily reduced with NADPH. SudA is highly homologous to KOD1-GO-GAT (or KOD1-GltA), a single-gene encoded protein in Pyrococcus kodakaraensis, which has been putatively identified as hyperthermophilic glutamate synthase. However, P. furiosus sulfide dehydrogenase does not have glutamate synthase activity. SudB is highly homologous to HydG, the gamma-subunit of P. furiosus NiFe hydrogenase. The latter enzyme also has sulfide dehydrogenase activity. The P. furiosus genome contains a second set of consecutive genes, sudY and sudX, with very high homology to the sudB and sudA genes, and possibly encoding a sulfide dehydrogenase isoenzyme. Each subunit of sulfide dehydrogenase is a primary structural paradigm for a different class of iron-sulfur flavoproteins.

Nitrogenase of Azotobacter vinelandii: kinetic analysis of the Fe protein redox cycle.

Nitrogenase consists of two metalloproteins (Fe protein and MoFe protein) which are assumed to associate and dissociate to transfer a single electron to the substrates. This cycle, called the Fe protein cycle, is driven by MgATP hydrolysis and is repeated until the substrates are completely reduced. The rate-limiting step of the cycle, and substrate reduction, is suggested to be the dissociation of the Fe protein-MoFe protein complex which is obligatory for the reduction of the Fe protein [Thorneley, R. N. F., and Lowe, D. J. (1983) Biochem. J. 215, 393-403]. This hypothesis is based on experiments with dithionite as the reductant. We also tested besides dithionite flavodoxin hydroquinone, a physiological reductant. Two models could describe the experimental data of the reduction by dithionite. The first model, with no reduction of Fe protein bound to MoFe protein, predicts a rate of dissociation of the protein complex of 8.1 s-1. This rate is too high to be the rate-limiting step of the Fe protein cycle (kobs = 3.0 s-1). The second model, with reduction of the Fe protein in the nitrogenase complex, predicts a rate of dissociation of the protein complex of 2.3 s-1, which in combination with reduction of the nitrogenase complex can account for the observed turnover rate of the Fe protein cycle. When flavodoxin hydroquinone (155 microM) was the reductant, the rate of reduction of oxidized Fe protein in the nitrogenase complex (kobs approximately 400 s-1) was 100 times faster than the turnover rate of the cycle with flavodoxin as the reductant (4 s-1). Pre-steady-state electron uptake experiments from flavodoxin hydroquinone indicate that before and after reduction of the nitrogenase complex relative slow reactions take place, which limits the rate of the Fe protein cycle. These results are discussed in the context of the kinetic models of the Fe protein cycle of nitrogenase.

A cytochrome cbb3 (cytochrome c) terminal oxidase in Azospirillum brasilense Sp7 supports microaerobic growth.

Spectral analysis indicated the presence of a cytochrome cbb3 oxidase under microaerobic conditions in Azospirillum brasilense Sp7 cells. The corresponding genes (cytNOQP) were isolated by using PCR. These genes are organized in an operon, preceded by a putative anaerobox. The phenotype of an A. brasilense cytN mutant was analyzed. Under aerobic conditions, the specific growth rate during exponential phase (mu(e)) of the A. brasilense cytN mutant was comparable to the wild-type specific growth rate (m(e) of approximately 0.2 h-1). In microaerobic NH4+-supplemented conditions, the low respiration of the A. brasilense cytN mutant affected its specific growth rate (mu(e) of approximately 0.02 h-1) compared to the wild-type specific growth rate (mu(e) of approximately 0.2 h-1). Under nitrogen-fixing conditions, both the growth rates and respiration of the wild type were significantly diminished in comparison to those under NH4+-supplemented conditions. Differences in growth rates and respiration between the wild type and the A. brasilense cytN mutant were less pronounced under these nitrogen-fixing conditions (mu(e) of approximately 0.03 h-1 for the wild type and 0.02 h-1 for the A. brasilense cytN mutant). The nitrogen-fixing capacity of the A. brasilense cytN mutant was still approximately 80% of that determined for the wild-type strain. This leads to the conclusion that the A. brasilense cytochrome cbb3 oxidase is required under microaerobic conditions, when a high respiration rate is needed, but that under nitrogen-fixing conditions the respiration rate does not seem to be a growth-limiting factor.

Redox properties and electron paramagnetic resonance spectroscopy of the transition state complex of Azotobacter vinelandii nitrogenase.

Nitrogenase is a two-component metalloenzyme that catalyzes a MgATP hydrolysis driven reduction of substrates. Aluminum fluoride plus MgADP inhibits nitrogenase by stabilizing an intermediate of the on-enzyme MgATP hydrolysis reaction. We report here the redox properties and electron paramagnetic resonance (EPR) signals of the aluminum fluoride-MgADP stabilized nitrogenase complex of Azotobacter vinelandii. Complex formation lowers the midpoint potential of the [4Fe-4S] cluster in the Fe protein. Also, the two-electron reaction of the unique [8Fe-7S] cluster in the MoFe protein is split in two one-electron reactions both with lower midpoint potentials. Furthermore, a change in spin-state of the two-electron oxidized [8Fe-7S] cluster is observed. The implications of these findings for the mechanism of MgATP hydrolysis driven electron transport within the nitrogenase protein complex are discussed.

Pre-steady-state kinetics of nitrogenase from Azotobacter vinelandii. Evidence for an ATP-induced conformational change of the nitrogenase complex as part of the reaction mechanism.

The pre-steady-state electron transfer reactions of nitrogenase from Azotobacter vinelandii have been studied by stopped-flow spectrophotometry. With reduced nitrogenase proteins after the initial absorbance increase at 430 nm (which is associated with electron transfer from the Fe protein to the MoFe protein and is complete in 50 ms) the absorbance decreases, which, dependent on the ratio [Av2]/[Av1], is followed by an increase of the absorbance. The mixing of reductant-free nitrogenase proteins with MgATP leads after 20 ms to a decrease of the absorbance, which could be fitted (from 0. 05 to 1 s) with a single exponential decay with a rate constant kobs = 6.6 +/- 0.8 s-1. This reaction of nitrogenase was measured at different wavelengths. The data indicate the formation of a species with a blue shift of the absorbance of metal-sulfur clusters of nitrogenase from 430 to 360 nm. The absorbance decrease at 430 nm observed (after 50 ms) in the case of the reduced nitrogenase proteins could only be simulated well if, after the initial electron transfer from the Fe protein to the MoFe protein and before dissociation of the nitrogenase complex, an additional reaction was assumed. The rate constant of this reaction was of the same order as the rate constant of the MgATP-dependent pre-steady-state proton production by nitrogenase from A. vinelandii: kobs = 14 +/- 4 s-1 with reduced nitrogenase proteins and kobs = 6 +/- 2 s-1 with dithionite-free nitrogenase proteins (Duyvis, M. G., Wassink, H., and Haaker, H. (1994) Eur. J. Biochem. 225, 881-890). It is proposed that in the presence and absence of reductant, the observed absorbance decrease at 430 nm of nitrogenase is caused by a change of the conformation of the nitrogenase complex, as a consequence of hydrolysis of MgATP.

Respiratory control determines respiration and nitrogenase activity of Rhizobium leguminosarum bacteroids.

The relationship between the O2 input rate into a suspension of Rhizobium leguminosarum bacteroids, the cellular ATP and ADP pools, and the whole-cell nitrogenase activity during L-malate oxidation has been studied. It was observed that inhibition of nitrogenase by excess O2 coincided with an increase of the cellular ATP/ADP ratio. When under this condition the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) was added, the cellular ATP/ADP ratio was lowered while nitrogenase regained activity. To explain these observations, the effects of nitrogenase activity and CCCP on the O2 consumption rate of R. leguminosarum bacteroids were determined. From 100 to 5 microM O2, a decline in the O2 consumption rate was observed to 50 to 70% of the maximal O2 consumption rate. A determination of the redox state of the cytochromes during an O2 consumption experiment indicated that at O2 concentrations above 5 microM, electron transport to the cytochromes was rate-limiting oxidation and not the reaction of reduced cytochromes with oxygen. The kinetic properties of the respiratory chain were determined from the deoxygenation of oxyglobins. In intact cells the maximal deoxygenation activity was stimulated by nitrogenase activity or CCCP. In isolated cytoplasmic membranes NADH oxidation was inhibited by respiratory control. The dehydrogenase activities of the respiratory chain were rate-limiting oxidation at O2 concentrations (if >300 nM. Below 300 nM the terminal oxidase system followed Michaelis-Menten kinetics (Km of 45 +/- 8 nM). We conclude that (i) respiration in R. leguminosarum bacteroids takes place via a respiratory chain terminating at a high-affinity oxidase system, (ii) the activity of the respiratory chain is inhibited by the proton motive force, and (iii) ATP hydrolysis by nitrogenase can partly relieve the inhibition of respiration by the proton motive force and thus stimulate respiration at nanomolar concentrations of O2.

Formation and characterization of a transition state complex of Azotobacter vinelandii nitrogenase.

A stable complex is formed between the nitrogenase proteins of Azotobacter vinelandii, aluminium fluoride and MgADP. All nitrogenase activities are inhibited. The complex formation was found to be reversible. An incubation at 50 degrees C recovers nitrogenase activity. The complex has been characterized with respect to protein and nucleotide composition and redox state of the metal-sulfur clusters. Based on the inhibition by aluminium fluoride together with MgADP, it is proposed that a stable transition state complex with nitrogenase is isolated.

Properties of the Peribacteroid Membrane ATPase of Pea Root Nodules and Its Effect on the Nitrogenase Activity.

Peribacteroid membrane vesicles from pea (Pisum sativum) root nodules were isolated from membrane-enclosed bacteroids by an osmotic shock. The ATPase activity associated with this membrane preparation was characterized, and its electrogenic properties were determined. The pH gradient was measured as a change of the fluorescence intensity of 9-amino-6-chloro-2-methoxyacridine and the membrane potential as a shift of absorbance of bis-(3-propyl-5-oxoisoxazol-4-yl)pentamethine oxonol. It was demonstrated that the ATPase generates a pH gradient as well as a membrane potential across the peribacteroid membrane. The reversibility of the ATPase was demonstrated by a light-dependent ATP synthesis by peribacteroid membrane vesicles fused with bacteriorhodopsin-phospholipid vesicles. The light-driven ATP synthesis by the peribacteroid membrane ATPase was completely inhibited by a proton-conducting ionophore. The proton-pumping activity of the peribacteroid membrane ATPase could also be demonstrated with peribacteroid membrane-enclosed bacteroids, and effects on nitrogenase activity were established. At pH values below 7.5, an active peribacteroid membrane ATPase inhibited the nitrogenase activity of peribacteroid membrane-enclosed bacteroids. At pH values above 8, at which whole cell nitrogenase activity was inhibited, the protonpumping activity of the peribacteroid membrane ATPase could partially reverse the pH inhibition. Vanadate, an inhibitor of plasma membrane and peribacteroid membrane ATPases, stimulated nodular nitrogenase activity. It will be proposed that the proton-pumping activity of the peribacteroid membrane ATPase in situ is a possible regulator of nodular nitrogenase activity.

Pre-steady-state MgATP-dependent proton production and electron transfer by nitrogenase from Azotobacter vinelandii.

MgATP-dependent pre-steady-state proton production by nitrogenase from Azotobacter vinelandii was studied by monitoring the absorbance changes at 572 nm of the pH indicator o-cresolsulphonphtalein in a weakly buffered solution. The absorbance changes are characterized by a constant phase, a single exponential decrease and a linear decrease. The observed rate constant for the single exponential MgATP-dependent proton production by reduced nitrogenase proteins at 20.0 degrees C is 14 +/- 4 s-1. No proton production with a rate constant comparable to the observed rate constant of electron transfer (kobs approximately 100 s-1) was detected. The extent of the observed MgATP-dependent proton production is determined by the redox state of the nitrogenase proteins before mixing with MgATP; less protons are produced when more electrons are transferred from the Fe protein to the MoFe protein. Values of 2.7 +/- 0.3 mol H+produced/mol MoFe protein with oxidized Fe protein, and 1.1 +/- 0.1 mol H+produced/mol MoFe protein with reduced Fe protein, were found. The data are interpreted to mean that protons are taken up after electron transfer from the Fe protein to the MoFe protein; the ratio electrons(transferred)/H-uptake was calculated to be 1.2 +/- 0.2. After mixing the nitrogenase proteins with MgADP, proton production takes place as well. The proton-production curve did not have a constant phase and the observed rate constant of the single exponential reaction is higher, compared to MgATP-dependent proton production (kobs approximately 35 s-1). The amount of protons produced depends also on the redox state of the Fe protein; no proton production was observed with the oxidized Fe protein; with dithionite-reduced Fe protein a value of 3.1 +/- 0.4 mol H+produced/mol MoFe protein was found (or 0.5 +/- 0.1 mol H+/mol Fe protein). Similar results were obtained when only the Fe protein was mixed with MgADP, but the observed absorbance changes were smaller; mixing of dithionite-reduced Fe protein with MgADP resulted in the production of 0.17 +/- 0.05 mol H+/mol Fe protein. All reported absorbance changes were absent when the experiments were performed in a buffered solution. The series of events that occur after mixing of the nitrogenase proteins with MgATP will be presented and discussed. In the case of the reduced Fe protein, electron transfer takes place at a rate of 100 s-1, which is followed by H+ production (kobs approximately 14 s-1). When there is no electron transfer (oxidized Fe protein) the rate constant of the MgATP-induced proton production decreases.(ABSTRACT TRUNCATED AT 400 WORDS)

Redox properties and EPR spectroscopy of the P clusters of Azotobacter vinelandii MoFe protein.

In Azotobacter vinelandii MoFe protein the oxidation of the P clusters to the S = 7/2 state is associated with a redox reaction with Em,7.5 = +90 +/- 10 mV (vs the normal hydrogen electrode), n = 1. A concomitant redox process is observed for a rhombic S = 1/2 EPR signal with g = 1.97, 1.88 and 1.68. This indicates that both S = 1/2 and S = 7/2 signals are associated with oxidized P clusters occurring as a physical mixture of spin states. The maximal intensity of the S = 1/2 and S = 7/2 signals in the mediated equilibrium redox titration is similar if not identical to that of solid-thionine-treated samples. Summation of the spin concentration of the S = 1/2 spin state (0.25 +/- 0.03 spin/alpha 2 beta 2) and the S = 7/2 spin state (1.3 +/- 0.2 spin/alpha 2 beta 2) confirms that the MoFe protein has absolutely no more than two P clusters. In spectra of enzyme fixed at potentials around -100 mV a very low-intensity g = 12 EPR signal was discovered. In parallel-mode EPR the signal sharpened and increased > 10-fold in intensity which allowed us to assign the g = 12 signal to a non-Kramers system (presumably S = 3). In contrast with the non-Kramers EPR signals of various metalloproteins and inorganic compounds, the sharp absorption-shaped g = 12 signal is not significantly broadened into zero field, implying that the zero field splitting of the non-Kramers doublet is smaller than the X-band microwave quantum. The temperature dependence of this g = 12 EPR signal indicates that it is from an excited state within the integer spin multiplet. A bell-shaped titration curve with Em,7.5 = -307 +/- 30 mV and +81 +/- 30 mV midpoint potentials is found for the g = 12 EPR signal. We propose that this signal represents an intermediate redox state of the P clusters between the diamagnetic, dithionite-reduced and the fully oxidized S = 7/2 and S = 1/2 state. Redox transitions of two electrons (-307 +/- 30 mV) and one electron (+90 +/- 10 mV) link the sequence S = 0<-->S = 3<-->(S = 7/2 and S = 1/2). We propose to name the latter paramagnetic oxidation states of the P clusters in nitrogenase POX1 and POX2, and to retain PN for the diamagnetic native redox state.(ABSTRACT TRUNCATED AT 400 WORDS)

Temperature effects on the MgATP-induced electron transfer between the nitrogenase proteins from Azotobacter vinelandii.

The temperature dependence of the pre-steady-state MgATP-dependent electron transfer from the MoFe protein to the Fe protein of the nitrogenase from Azotobacter vinelandii has been investigated between 6 degrees C and 31 degrees C by stopped-flow spectrophotometry. Below 14 degrees C, the data are consistent with a model in which interaction of MgATP with nitrogenase is fast and irreversible, and is followed by reversible electron transfer. From the extent and from the rate of the absorbance change, the rate constants for electron transfer from Fe protein to MoFe protein and of the reverse reaction were calculated. The direct rate constant increases with temperature (6-14 degrees C) from about 1 s-1 to about 26 s-1. The rate constant for the reverse reaction was found to be approximately 4 s-1 and invariant with the reaction temperature. Analysis of the data obtained in the temperature range between 6 degrees C and 12 degrees C within the framework of the transition-state theory show that electron transfer from the Fe protein to the MoFe protein occurs via a highly disordered transition state with activation parameters delta H(0) ++ = 289 kJ.mol-1 and delta S(0) ++ = 792 J.K-1.mol-1. The Eyring plot of the stopped-flow data displays an inflection point around 14 degrees C. From the stopped-flow data obtained between 18 degrees and 27 degrees C the activation parameters delta H(0) ++ and delta S(0) ++ for the reduction of the MoFe protein by Fe protein are calculated to be 90 kJ.mol-1 and 99 J.K-1.mol-1 respectively. A second inflection point in the Eyring plot could exist around 28 degrees C.

A reinvestigation of the pre-steady-state ATPase activity of the nitrogenase from Azotobacter vinelandii.

The pre-steady-state ATPase activity of nitrogenase has been reinvestigated. The exceptionally high burst in the hydrolysis of MgATP by the nitrogenase from Azotobacter vinelandii communicated by Cordewener et al. (1987) [Cordewener J., ten Asbroek A., Wassink H., Eady R. R., Haaker H. & Veeger C. (1987) Eur. J. Biochem. 162, 265-270] was found to be caused by an apparatus artefact. A second possible artefact in the determination of the stoichiometry of the pre-steady-state ATPase activity of nitrogenase was observed. Acid-quenched mixtures of dithionite-reduced MoFe or Fe protein of Azotobacter vinelandii nitrogenase and MgATP contained phosphate above the background level. It is proposed that due to this reaction, quenched reaction mixtures of nitrogenase and MgATP may contain phosphate in addition to the phosphate released by the ATPase activity of the nitrogenase complex. It was feasible to monitor MgATP-dependent pre-steady-state proton production by the absorbance change at 572 nm of the pH indicator o-cresolsulfonaphthalein in a weakly buffered solution. At 5.6 degrees C, a pre-steady-state phase of H+ production was observed, with a first-order rate constant of 2.2 s-1, whereas electron transfer occurred with a first-order rate constant of 4.9 s-1. At 20.0 degrees C, MgATP-dependent H+ production and electron transfer in the pre-steady-state phase were characterized by observed rate constants of 9.4 s-1 and 104 s-1, respectively. The stopped-flow technique failed to detect a burst in the release of protons by the dye-oxidized nitrogenase complex. It is concluded that the hydrolysis rate of MgATP, as judged by proton release, is lower than the rate of electron transfer from the Fe protein to the MoFe protein.

Glutamate Oxaloacetate Transaminase in Pea Root Nodules : Participation in a Malate/Aspartate Shuttle between Plant and Bacteroid.

Glutamate oxaloacetate transaminase (l-glutamate: oxaloacetate aminotransferase, EC 2.6.1.1 [GOT]), a key enzyme in the flow of carbon between the organic acid and amino acid pools in pea (Pisum sativum L.) root nodules, was studied. By ion exchange chromatography, the presence of two forms of GOT in the cytoplasm of pea root nodule cells was established. The major root nodule form was present in only a small quantity in the cytoplasm of root cells. Fractionation of root nodule cell extracts demonstrated that the increase in the GOT activity during nodule development was due to the increase of the activity in the cytoplasm of the plant cells, and not to an increase in activity in the plastids or in the mitochondria. The kinetic properties of the different cytoplasmic forms of GOT were studied. Some of the K(m) values differed, but calculations indicated that not the kinetic properties but a high concentration of the major root nodule form caused the observed increase in GOT activity in the pea root nodules. It was found that the reactions of the malate/aspartate shuttle are catalyzed by intact bacteroids, and that these reactions can support nitrogen fixation. It is proposed that the main function of the nodule-stimulated cytoplasmic form of GOT is participation in this shuttle.

The role of MgATP hydrolysis in nitrogenase catalysis.

Kinetic studies on MgATP hydrolysis by nitrogenase of Azotobacter vinelandii were performed in the presence and in the absence of reducing equivalents. By measuring the ATPase activity of dye-oxidized nitrogenase proteins it can be excluded that reductant-independent ATPase activity is the result of futile cycling of electrons. The turnover rates of MoFe protein during reductant-dependent and reductant-independent ATPase activity, when measured with excess Fe protein, have approximately the same value, i.e. 5 s-1 at pH 7.4 and 22 degrees C, assuming the hydrolysis of four molecules of MgATP per turnover of MoFe protein. For Fe protein on the other hand, the maximum turnover rate during reductant-independent ATPase activity is only about 6% of that of reductant-dependent ATPase activity. While the reductant-dependent ATPase activity shows a sigmoidal dependence on the concentration of MgATP, the reductant-independent ATPase activity yields hyperbolic saturation curves. To account for these results it is proposed that the rate-limiting step during MgATP hydrolysis by oxidized nitrogenase is the rate of regeneration of active Fe protein. In the presence of reductant, the regeneration of active Fe protein is stimulated, explaining the higher ATPase activity of nitrogenase during substrate reduction.

Identification of cytoplasmic nodule-associated forms of malate dehydrogenase involved in the symbiosis between Rhizobium leguminosarum and Pisum sativum.

The malate dehydrogenase activity (EC 1.1.1.37), present in the cytoplasm of Pisum sativum root nodules, can be separated by ion-exchange chromatography into four different fractions. Malate dehydrogenase activity present in the cytoplasm of roots elutes mainly as a single peak. During nodule development an increase in malate dehydrogenase activity per gram of material was observed. This increase occurred concomitantly with the increase in nitrogenase activity. The kinetic properties of the separated malate dehydrogenases of root nodule cytoplasm and root cytoplasm were studied. The Km values for malate (2.6 mM), NAD+ (27 microM), oxaloacetate (18 microM) and NADH (13 microM) of the dominant form of the root nodule cytoplasm are much lower than those of the dominant malate dehydrogenase root form (64 mM, 4.4 mM, 89 microM and 70 microM respectively). Binding of malate by the enzyme-NADH complex from root nodules results in an abortive complex, thereby blocking the further reduction of oxaloacetate by NADH. The dominant root malate dehydrogenase does not form the abortive complex. From the kinetic data it is concluded, first, that the root nodule forms of the enzyme are capable of catalysing at a high rate the reduction of oxaloacetate, to meet the demands for malate governed by the bacteroid and the infected plant cell. The second conclusion, drawn from the kinetic data, is that under physiological conditions the conversion of oxaloacetate can be controlled just by the malate concentration. Consequently the major root nodule forms of malate dehydrogenase are able to allow a high flux of malate production from oxaloacetate but also to establish a sufficient oxaloacetate concentration necessary for the assimilation and transport of fixed nitrogen.

Quantitative EPR of an S = 7/2 system in thionine-oxidized MoFe proteins of nitrogenase. A redefinition of the P-cluster concept.

Thionine-oxidized nitrogenase MoFe proteins from Azotobacter vinelandii. Azotobacter chroococcum and Klebsiella pneumoniae exhibit excited-state EPR signals with g = 10.4, 5.8 and 5.5 with a maximal amplitude in the temperature range of 20-50 K. The magnitude of these effective g values, combined with the temperature dependence of the peak area at g = 10.4 from 12 K to 86 K, are consistent with an S = 7/2 system with spin Hamiltonian parameters D = -3.7 +/- 0.7 cm-1, [E] = 0.16 +/- 0.01 cm-1 and g = 2.00. This interpretation predicts nine additional effective g values some of which have been detected as broad features of low intensity at g approximately 10, approximately 2.5 and approximately 1.8. The S = 7/2 EPR is ascribed to the multi-iron exchange-coupled entities known as the P clusters. Quantification relative to the S = 3/2 EPR signal from dithionite-reduced MoFe protein indicates a stoichiometry of one P cluster per FeMo cofactor. Two possible interpretations for these observations, together with data from the literature, are proposed. In the first model there are two P clusters per tetrameric MoFe protein. Each P cluster encompasses approximately 8Fe ions and releases a total of three electrons on oxidation with excess thionine. In the second model the conventional view of four P clusters, each containing approximately 4Fe, is retained. This alternative requires that following one-electron oxidation, the P clusters factorize into two populations, Pa and Pb, only one of which is further oxidized with thionine resulting in the S = 7/2 system. Both models require eight-electron oxidation of tetrameric MoFe protein to reach the S = 7/2 state.

Binding of ADP and orthophosphate during the ATPase reaction of nitrogenase.

The pre-steady-state ATPase activity of nitrogenase from Azotobacter vinelandii was investigated. By using a rapid-quench technique, it has been demonstrated that with the oxidized nitrogenase complex the same burst reaction of MgATP hydrolysis occurs as observed with the reduced complex, namely 6-8 mol orthophosphate released/mol MoFe protein. It is concluded that the pre-steady-state ATPase activity is independent of electron transfer from Fe protein to MoFe protein. Results obtained from gel centrifugation experiments showed that during the steady state of reductant-independent ATP hydrolysis there is a slow dissociation of one molecule of MgADP from the nitrogenase proteins (koff less than or equal to 0.2 s-1); the second MgADP molecule dissociates much faster (koff greater than or equal to 0.6 s-1). Under the same conditions orthophosphate was found to be associated with the nitrogenase proteins. The rate of dissociation of orthophosphate from the nitrogenase complex, as estimated from the gel centrifugation experiments, is in the same order of magnitude as the steady-state turnover rate of the reductant-independent ATPase activity (0.6 mol Pi formed X s-1 X mol Av2(-1) at 22 degrees C). These data are consistent with dissociation of orthophosphate or MgADP being rate-limiting during nitrogenase-catalyzed reductant-independent ATP hydrolysis.

Characterization of three different flavodoxins from Azotobacter vinelandii.

The flavodoxins from Azotobacter vinelandii cells grown N2-fixing and from cells grown on NH4OAc have been purified and characterized. The purified flavodoxins from these cells are a mixture of three different flavodoxins (Fld I, II, III) with different primary structures. The three proteins were separated by fast protein liquid chromatography; Fld I eluted at 0.38 M KCl, Fld II at 0.43 M KCl and Fld III at 0.45 M KCl. The most striking difference between the three flavodoxins was the midpoint potential (pH 7.0, 25 degrees C) of the semiquinone/hydroquinone couple, which was -320 mV for Fld I and -500 mV for the other two flavodoxins (Fld II and Fld III). All three flavodoxins were present in cells grown on NH4OAc. In cells grown on N2 as N source only Fld I and Fld II were found. The concentration of Fld II was 10-fold higher in N2-fixing cells than in cells grown on NH4OAc. Evidence has been obtained that Fld II is involved in electron transport to nitrogenase. As will be discussed, our observation that preparations of Azotobacter flavodoxin are heterogeneous, has consequences for the published data.

Studies on the mechanism of electron transport to nitrogenase in Azotobacter vinelandii.

The involvement of the cytoplasmic membrane in electron transport to nitrogenase has been studied. Evidence shows that nitrogenase activity in Azotobacter vinelandii is coupled to the flux of electrons through the respiratory chain. To obtain information about proteins involved, the changes occurring in A. vinelandii cells transferred to nitrogen-free medium after growth on NH4Cl (depression of nitrogenase activity) were studied. Synthesis of the nitrogenase polypeptides was detectable 5 min after transfer to nitrogen-free medium. No nitrogenase activity could be detected until t = 20 min, whereupon a linear increase of nitrogenase activity with time was observed. Synthesis of nitrogenase was accompanied by synthesis of flavodoxin II and two membrane-bound polypeptides of Mr 29,000 and 30,000. Analysis with respect to changes in membrane-bound NAD(P)H dehydrogenase activities revealed the induction of an NADPH dehydrogenase activity, which was not detectable in membranes isolated from cells grown in the presence of NH4OAc. This induced activity was associated with the appearance of a polypeptide of Mr 29,000 in the NADPH dehydrogenase complex.